Neuropilin-1 regulates renin synthesis in juxtaglomerular cells.

Renin is the key enzyme of the systemic renin-angiotensin-aldosterone system, which plays an essential role in regulating blood pressure and maintaining electrolyte and extracellular volume homeostasis. Renin is mainly produced and secreted by specialized juxtaglomerular (JG) cells in the kidney. In the present study, we report for the first time that the conserved transmembrane receptor neuropilin-1 (NRP1) participates in the development of JG cells and plays a key role in renin production. We used the myelin protein zero-Cre (P0-Cre) to abrogate Nrp1 constitutively in P0-Cre lineage-labelled cells of the kidney. We found that the P0-Cre precursor cells differentiate into renin-producing JG cells. We employed a lineage-tracing strategy combined with RNAscope quantification and metabolic studies to reveal a cell-autonomous role for NRP1 in JG cell function. Nrp1-deficient animals displayed abnormal levels of tissue renin expression and failed to adapt properly to a homeostatic challenge to sodium balance. These findings provide new insights into cell fate decisions and cellular plasticity operating in P0-Cre-expressing precursors and identify NRP1 as a novel key regulator of JG cell maturation. KEY POINTS: Renin is a centrepiece of the renin-angiotensin-aldosterone system and is produced by specialized juxtaglomerular cells (JG) of the kidney. Neuropilin-1 (NRP1) is a conserved membrane-bound receptor that regulates vascular and neuronal development, cancer aggressiveness and fibrosis progression. We used conditional mutagenesis and lineage tracing to show that NRP1 is expressed in JG cells where it regulates their function. Cell-specific Nrp1 knockout mice present with renin paucity in JG cells and struggle to adapt to a homeostatic challenge to sodium balance. The results support the versatility of renin-producing cells in the kidney and may open new avenues for therapeutic approaches.

Biallelic CAV1 null variants induce congenital generalized lipodystrophy with achalasia.

OBJECTIVE: CAV1 encodes caveolin-1, a major protein of plasma membrane microdomains called caveolae, involved in several signaling pathways. Caveolin-1 is also located at the adipocyte lipid droplet. Heterozygous pathogenic variants of CAV1 induce rare heterogeneous disorders including pulmonary arterial hypertension and neonatal progeroid syndrome. Only one patient was previously reported with a CAV1 homozygous pathogenic variant, associated with congenital generalized lipodystrophy (CGL3). We aimed to further delineate genetic transmission, clinical, metabolic, and cellular characteristics of CGL3. DESIGN/METHODS: In a large consanguineous kindred referred for CGL, we performed next-generation sequencing, as well as clinical, imagery, and metabolic investigations. We studied skin fibroblasts from the index case and the previously reported patient with CGL3. RESULTS: Four patients, aged 8 months to 18 years, carried a new homozygous p.(His79Glnfs*3) CAV1 variant. They all displayed generalized lipodystrophy since infancy, insulin resistance, low HDL-cholesterol, and/or high triglycerides, but no pulmonary hypertension. Two patients also presented at the age of 15 and 18 years with dysphagia due to achalasia, and one patient had retinitis pigmentosa. Heterozygous parents and relatives (n = 9) were asymptomatic, without any metabolic abnormality. Patients' fibroblasts showed a complete loss of caveolae and no protein expression of caveolin-1 and its caveolin-2 and cavin-1 partners. Patients' fibroblasts also displayed insulin resistance, increased oxidative stress, and premature senescence. CONCLUSIONS: The CAV1 null variant investigated herein leads to an autosomal recessive congenital lipodystrophy syndrome. Loss of caveolin-1 and/or caveolae induces specific manifestations including achalasia which requires specific management. Overlapping phenotypic traits between the different CAV1-related diseases require further studies.

The spectrum of kidney biopsies in hospitalized patients with COVID-19, acute kidney injury, and/or proteinuria.

We report a multicentric retrospective case series of patients with COVID-19 who developed acute kidney injury and/or proteinuria and underwent a kidney biopsy in the Paris and its metropolitan area. Forty-seven patients (80.9% men) with COVID-19 who underwent a kidney biopsy between March 08 and May 19, 2020 were included. Median age was 63 years IQR [52-69]. Comorbidities included hypertension (66.0%), diabetes mellitus (27.7%), obesity (27.7%), history of chronic kidney (25.5%), cardiac (38.6%) and respiratory (27.3%) diseases. Initial symptoms were fever (85.1%), cough (63.8%), shortness of breath (55.3%), and diarrhea (23.4%). Almost all patients developed acute kidney injury (97.9%) and 63.8% required renal replacement therapy. Kidney biopsy showed two main histopathological patterns, including acute tubular injury in 20 (42.6%) patients, and glomerular injury consisting of collapsing glomerulopathy and focal segmental glomerulosclerosis in 17 (36.2%) patients. Two (4.3%) patients had acute vascular nephropathy, while eight (17%) had alternative diagnosis most likely unrelated to COVID-19. Acute tubular injury occurred almost invariably in the setting of severe forms of COVID-19, whereas patients with glomerular injury had various profiles of COVID-19 severity and collapsing glomerulopathy was only observed in patients harboring a combination of APOL1 risk variants. At last follow-up, 16 of the 30 patients who initially required dialysis were still on dialysis, and 9 died. The present study describes the spectrum of kidney lesions in patients with COVID-19. While acute tubular injury is correlated with COVID-19 severity, the pattern of glomerular injury is intimately associated with the expression of APOL1 risk variants.

De Novo Focal and Segmental Glomerulosclerosis After COVID-19 in a Patient With a Transplanted Kidney From a Donor With a High-risk APOL1 Variant.

BACKGROUND: There is compelling evidence that renal complications in a native kidney are a major concern in patients infected with severe acute respiratory syndrome coronavirus 2, the causal agent of coronavirus disease 2019 (COVID-19). The spectrum of renal lesions observed on renal grafts in this context remains to be determined. METHODS: We report the case of a renal transplant recipient with non-severe COVID-19, who subsequently developed nephrotic syndrome associated with acute renal injury. RESULTS: Renal biopsy demonstrated focal and segmental glomerulosclerosis lesions classified as not otherwise specified histological variant. Genotyping for 2 risk alleles of the apolipoprotein L1 gene demonstrated that the donor was homozygous for the G2/G2 genotype. CONCLUSIONS: In renal transplant patients receiving kidneys from donors with high-risk apolipoprotein L1 variants, COVID-19 may promote acute glomerular injury in the form of focal and segmental glomerulosclerosis.

Vitamin D and Calcium Supplementation Accelerates Randall's Plaque Formation in a Murine Model.

Most kidney stones are made of calcium oxalate crystals. Randall's plaque, an apatite deposit at the tip of the renal papilla, is considered to at the origin of these stones. Hypercalciuria may promote Randall's plaque formation and growth. We analyzed whether long-term exposure of Abcc6-/- mice (a murine model of Randall's plaque) to vitamin D supplementation, with or without a calcium-rich diet, would accelerate the formation of Randall's plaque. Eight groups of mice (including Abcc6-/- and wild type) received vitamin D alone (100,000 UI/kg every 2 weeks), a calcium-enriched diet alone (calcium gluconate 2 g/L in drinking water), both vitamin D supplementation and a calcium-rich diet, or a standard diet (controls) for 6 months. Kidney calcifications were assessed by 3-dimensional microcomputed tomography, µ-Fourier transform infrared spectroscopy, field emission-scanning electron microscopy, transmission electron microscopy, and Yasue staining. At 6 months, Abcc6-/- mice exposed to vitamin D and calcium supplementation developed massive Randall's plaque when compared with control Abcc6-/- mice (P < 0.01). Wild-type animals did not develop significant calcifications when exposed to vitamin D. Combined administration of vitamin D and calcium significantly accelerates Randall's plaque formation in a murine model. This original model raises concerns about the cumulative risk of vitamin D supplementation and calcium intakes in Randall's plaque formation.

MFN2-associated lipomatosis: Clinical spectrum and impact on adipose tissue.

BACKGROUND: Multiple symmetric lipomatosis (MSL) is characterized by upper-body lipomatous masses frequently associated with metabolic and neurological signs. MFN2 pathogenic variants were recently implicated in a very rare autosomal recessive form of MSL. MFN2 encodes mitofusin-2, a mitochondrial fusion protein previously involved in Charcot-Marie-Tooth neuropathy. OBJECTIVE: To investigate the clinical, metabolic, tissular, and molecular characteristics of MFN2-associated MSL. METHODS: We sequenced MFN2 in 66 patients referred for altered fat distribution with one or several lipomas or lipoma-like regions and performed clinical and metabolic investigations in patients with positive genetic testing. Lipomatous tissues were studied in 3 patients. RESULTS: Six patients from 5 families carried a homozygous p.Arg707Trp pathogenic variant, representing the largest reported series of MFN2-associated MSL. Patients presented both lipomatous masses and a lipodystrophic syndrome (lipoatrophy, low leptinemia and adiponectinemia, hypertriglyceridemia, insulin resistance and/or diabetes). Charcot-Marie-Tooth neuropathy was of highly variable clinical severity. Lipomatous tissue mainly contained hyperplastic unilocular adipocytes, with few multilocular cells. It displayed numerous mitochondrial alterations (increased number and size, structural defects). As compared to control subcutaneous fat, mRNA and protein expression of leptin and adiponectin was strikingly decreased, whereas the CITED1 and fibroblast growth factor 21 (FGF21) thermogenic markers were strongly overexpressed. Consistently, serum FGF21 was markedly increased, and 18F-FDG-PET-scan revealed increased fat metabolic activity. CONCLUSION: MFN2-related MSL is a novel mitochondrial lipodystrophic syndrome involving both lipomatous masses and lipoatrophy. Its complex neurological and metabolic phenotype justifies careful clinical evaluation and multidisciplinary care. Low leptinemia and adiponectinemia, high serum FGF21, and increased 18F-FDG body fat uptake may be disease markers.

ABCC6 Deficiency Promotes Development of Randall Plaque.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a genetic disease caused by mutations in the ABCC6 gene that result in low pyrophosphate levels and subsequent progressive soft tissue calcifications. PXE mainly affects the skin, retina, and arteries. However, many patients with PXE experience kidney stones. We determined the prevalence of this pathology in patients with PXE and examined the possible underlying mechanisms in murine models. METHODS: We conducted a retrospective study in a large cohort of patients with PXE and analyzed urine samples and kidneys from Abcc6-/- mice at various ages. We used Yasue staining, scanning electron microscopy, electron microscopy coupled to electron energy loss spectroscopy, and Fourier transform infrared microspectroscopy to characterize kidney calcifications. RESULTS: Among 113 patients with PXE, 45 (40%) had a past medical history of kidney stones. Five of six computed tomography scans performed showed evidence of massive papillary calcifications (Randall plaques). Abcc6-/- mice spontaneously developed kidney interstitial apatite calcifications with aging. These calcifications appeared specifically at the tip of the papilla and formed Randall plaques similar to those observed in human kidneys. Compared with controls, Abcc6-/- mice had low urinary excretion of pyrophosphate. CONCLUSIONS: The frequency of kidney stones and probably, Randall plaque is extremely high in patients with PXE, and Abcc6-/- mice provide a new and useful model in which to study Randall plaque formation. Our findings also suggest that pyrophosphate administration should be evaluated for the prevention of Randall plaque and kidney stones.

Vancomycin-Associated Cast Nephropathy.

Vancomycin is a widely prescribed antibiotic, but the exact nature of vancomycin-associated nephrotoxicity is unclear, in particular when considering the frequent coadministration of aminoglycosides. We describe here the initial case of a 56-year-old woman with normal renal function developing unexplained ARF without hypovolemia after administration of vancomycin without coadministration of aminoglycosides. Studying the patient's renal biopsy specimen, we ascertained that obstructive tubular casts composed of noncrystal nanospheric vancomycin aggregates entangled with uromodulin explained the vancomycin-associated ARF. We developed in parallel a new immunohistologic staining technique to detect vancomycin in renal tissue and confirmed retrospectively that deleterious vancomycin-associated casts existed in eight additional patients with acute tubular necrosis in the absence of hypovolemia. Concomitant high vancomycin trough plasma levels had been observed in each patient. We also reproduced experimentally the toxic and obstructive nature of vancomycin-associated cast nephropathy in mice, which we detected using different in vivo imaging techniques. In conclusion, the interaction of uromodulin with nanospheric vancomycin aggregates represents a new mode of tubular cast formation, revealing the hitherto unsuspected mechanism of vancomycin-associated renal injury.

Topography, Composition and Structure of Incipient Randall Plaque at the Nanoscale Level.

PURPOSE: Randall identified calcium phosphate plaques in renal papillae as the origin of kidney stones. However, little is known about the early steps of Randall plaque formation preceding the onset of urolithiasis. Our objective was to characterize the composition and the initial formation site of incipient Randall plaque in nonstone forming, living patients. MATERIALS AND METHODS: Median patient age was 67.7 years. A total of 54 healthy papillae from kidneys removed for cancer and without stones were analyzed by immunohistochemistry and von Kossa staining, field emission-scanning electron microscopy with energy dispersive x-ray analysis, µ-Fourier transform infrared spectroscopy, cryo-transmission electron microscopy coupled to selected area electron diffraction and electron energy loss spectroscopy. RESULTS: Incipient Randall plaque was observed in 72.7% of kidneys. As expected, carbonated apatite was the main component of microcalcifications but amorphous calcium phosphate and whitlockite were identified in 80% and 40% of papillae, respectively. Incipient plaques were noted in the deepest part of the papillae around the loop of Henle tip as well as around the vasa recta, representing 62.4% and 37.2% of microcalcifications, respectively. Plaques were rarely close to collecting ducts. At the nanoscale level incipient calcifications were often composed of several nanocrystals in organic material that looked like microvesicles. CONCLUSIONS: Incipient Randall plaque is frequent. It appears not only at the extreme tip of the renal papillae around the hairpin structure of the loop of Henle but also around the vasa recta. Nanoscale analyses suggest a local nucleation process promoting nanocrystal growth in a supersaturated milieu. In addition, plaques contain various calcium and magnesium phosphates, and not only carbonated apatite.

HANAC Syndrome Col4a1 Mutation Causes Neonate Glomerular Hyperpermeability and Adult Glomerulocystic Kidney Disease.

Hereditary angiopathy, nephropathy, aneurysms, and muscle cramps (HANAC) syndrome is an autosomal dominant syndrome caused by mutations in COL4A1 that encodes the α1 chain of collagen IV, a major component of basement membranes. Patients present with cerebral small vessel disease, retinal tortuosity, muscle cramps, and kidney disease consisting of multiple renal cysts, chronic kidney failure, and sometimes hematuria. Mutations producing HANAC syndrome localize within the integrin binding site containing CB3[IV] fragment of the COL4A1 protein. To investigate the pathophysiology of HANAC syndrome, we generated mice harboring the Col4a1 p.Gly498Val mutation identified in a family with the syndrome. Col4a1 G498V mutation resulted in delayed glomerulogenesis and podocyte differentiation without reduction of nephron number, causing albuminuria and hematuria in newborns. The glomerular defects resolved within the first month, but glomerular cysts developed in 3-month-old mutant mice. Abnormal structure of Bowman's capsule was associated with metalloproteinase induction and activation of the glomerular parietal epithelial cells that abnormally expressed CD44,α-SMA, ILK, and DDR1. Inflammatory infiltrates were observed around glomeruli and arterioles. Homozygous Col4a1 G498V mutant mice additionally showed dysmorphic papillae and urinary concentration defects. These results reveal a developmental role for the α1α1α2 collagen IV molecule in the embryonic glomerular basement membrane, affecting podocyte differentiation. The observed association between molecular alteration of the collagenous network in Bowman's capsule of the mature kidney and activation of parietal epithelial cells, matrix remodeling, and inflammation may account for glomerular cyst development and CKD in patients with COL4A1-related disorders.

Spectrum and Prognosis of Noninfectious Renal Mixed Cryoglobulinemic GN.

Noninfectious mixed cryoglobulinemic GN (MCGN) has been poorly investigated. We analyzed presentation and outcome of 80 patients with biopsy-proven MCGN, which were identified in the retrospective French CryoVas survey. MCGN was related to primary Sjögren's syndrome in 22.5% of patients and to lymphoproliferative disorders in 28.7% of patients, and was defined as essential in 48.8% of patients. At presentation, hematuria, proteinuria ≥1 g/d, hypertension, and renal failure were observed in 97.4%, 84.8%, 85.3%, and 82.3% of cases, respectively. Mean±eGFR was 39.5±20.4 ml/min per 1.73 m(2) Membranoproliferative GN was the predominant histologic pattern, observed in 89.6% of cases. Renal interstitium inflammatory infiltrates were observed in 50% of cases. First-line treatment consisted of steroids alone (27.6%) or in association with rituximab (21.1%), alkylating agents (36.8%) or a combination of cyclophosphamide and rituximab (10.5%). After a mean follow-up of 49.9±45.5 months, 42.7% of patients relapsed with a renal flare in 75% of cases. At last follow-up, mean eGFR was 50.2±26.1 ml/min per 1.73 m(2)with 9% of patients having reached ESRD; 59% and 50% of patients achieved complete clinical and renal remission, respectively. A rituximab+steroids regimen prevented relapses more effectively than steroids alone or a cyclophosphamide+steroids combination did, but was associated with a higher rate of early death when used as first-line therapy. Severe infections and new-onset B-cell lymphoma occurred in 29.1% and 8.9% of cases, respectively; 24% of patients died. In conclusion, noninfectious MCGN has a poor long-term outcome with severe infections as the main cause of death.

Pseudo-Peritoneal Carcinomatosis Presentation of a Crystal-Storing Histiocytosis With an Unmutated Monoclonal κ Light Chain.

Crystal-storing histiocytosis (CSH) is a rare complication of monoclonal gammopathies caused by accumulation of crystalline material inside macrophages, and it may result in a variety of clinical manifestations depending on the involved organs. Although immunoglobulin κ light chains (LCs) seem to be the most frequent pathogenic component, very few molecular data are currently available.A 69-year-old man presented with a very poor performance status. Remarkable features were mesenteric lymph node enlargement and proteinuria, including a monoclonal κ LC. Light and electron microscopy studies revealed the presence of crystals within macrophages in the lymph nodes, bone marrow, and kidney, leading to the diagnosis of CSH. The pathogenic κ LC variable domain sequence was identical to the germline Vk3-2001/Jk201 gene segments, without any somatic mutation, suggesting an extra-follicular B cell proliferation.The patient was successfully treated with 4 cycles of bortezomib and dexamethasone. After a 12-month follow-up, he remains in hematological and renal remission.CSH may present as pseudo-peritoneal carcinomatosis and relate to a monoclonal κ LC encoded by an unmutated gene. Bortezomib-based therapy proved efficacious in this case.

Complete remission of monoclonal gammopathy with ocular and periorbital crystal storing histiocytosis and Fanconi syndrome.

A 62-year-old woman presented with crystalline keratopathy, crystal-storing histiocytosis, Fanconi syndrome, and a serum monoclonal IgG-κ and urinary κ light chain. Histology and electron microscopy studies revealed the presence of crystals within macrophages in multiple eye sites, in the kidney and in the bone marrow. The variable domain of the pathogenic κ light chain related to the Vk1-39 gene that was also involved in most previously reported cases of Fanconi syndrome. Owing to the severity of the damage to the eye and a potentially poor kidney prognosis, the patient underwent autologous stem cell transplantation. After 18 months follow-up, she is in complete hematological, ophthalmological, and renal remission.

LMNA mutations induce a non-inflammatory fibrosis and a brown fat-like dystrophy of enlarged cervical adipose tissue.

Some LMNA mutations responsible for insulin-resistant lipodystrophic syndromes are associated with peripheral subcutaneous lipoatrophy and faciocervical fat accumulation. Their pathophysiologic characteristics are unknown. We compared histologic, immunohistologic, ultrastructural, and protein expression features of enlarged cervical subcutaneous adipose tissue (scAT) obtained during plastic surgery from four patients with LMNA p.R482W, p.R439C, or p.H506D mutations versus cervical fat from eight control subjects, buffalo humps from five patients with HIV infection treated or not with protease inhibitors, and dorsocervical lipomas from two patients with mitochondrial DNA mutations. LMNA-mutated cervical scAT and HIV-related buffalo humps were dystrophic, with an increased percentage of small adipocytes, increased fibrosis without inflammatory features, and decreased number of blood vessels, as compared with control samples. Samples from patients with LMNA mutations or protease inhibitor-based therapy demonstrated accumulation of prelamin A, altered expression of adipogenic proteins and brown fat-like features, with an increased number of mitochondria and overexpression of uncoupling protein 1 (UCP1). These features were absent in samples from control subjects and from patients with HIV not treated with protease inhibitors. Mitochondrial DNA-mutated cervical lipomas demonstrated inflammatory fibrosis with distinct mitochondrial abnormalities but neither UCP1 expression nor prelamin A accumulation. In conclusion, Enlarged cervical scAT from patients with lipodystrophy demonstrated small adipocytes, fibrosis, and decreased vessel numbers. However, only cervical fat from patients with LMNA mutations or who had received protease inhibitor therapy accumulated prelamin A and exhibited similar remodeling toward a brown-like phenotype with UCP1 overexpression and mitochondrial alterations.

COL4A1 mutations and hereditary angiopathy, nephropathy, aneurysms, and muscle cramps.

BACKGROUND: COL4A3, COL4A4, and COL4A5 are the only collagen genes that have been implicated in inherited nephropathies in humans. However, the causative genes for a number of hereditary multicystic kidney diseases, myopathies with cramps, and heritable intracranial aneurysms remain unknown. METHODS: We characterized the renal and extrarenal phenotypes of subjects from three families who had an autosomal dominant hereditary angiopathy with nephropathy, aneurysms, and muscle cramps (HANAC), which we propose is a syndrome. Linkage studies involving microsatellite markers flanking the COL4A1-COL4A2 locus were performed, followed by sequence analysis of COL4A1 complementary DNA extracted from skin-fibroblast specimens from the subjects. RESULTS: We identified three closely located glycine mutations in exons 24 and 25 of the gene COL4A1, which encodes procollagen type IV alpha1. The clinical renal manifestations of the HANAC syndrome in these families include hematuria and bilateral, large cysts. Histologic analysis revealed complex basement-membrane defects in kidney and skin. The systemic angiopathy of the HANAC syndrome appears to affect both small vessels and large arteries. CONCLUSIONS: COL4A1 may be a candidate gene in unexplained familial syndromes with autosomal dominant hematuria, cystic kidney disease, intracranial aneurysms, and muscle cramps.

A humanized mouse model of idiopathic nephrotic syndrome suggests a pathogenic role for immature cells.

Idiopathic nephrotic syndrome is characterized by glomerular proteinuria in the absence of infiltrating cells or immunoglobulin deposits. Although it is suspected that T cells secrete a circulating factor that leads to proteinuria by altering the permeability of the glomerular filtration barrier, the precise etiology of this syndrome is unknown. Because an animal model that mimics human idiopathic nephrotic syndrome does not exist, we developed a humanized mouse model of the disease by injecting CD34(+) stem cells or CD34(-) peripheral blood mononuclear cells from afflicted patients into immunocompromised mice. Even though both CD34(+) and CD34(-) cells induced the engraftment of human CD45(+) leukocytes in mice, only the injection of CD34(+) stem cells induced albuminuria. Ultrastructural analysis of glomeruli from the resulting proteinuric mice revealed effacement of podocyte foot processes, similar to the pathology observed in the human disease. Therefore, our data suggest that the cells responsible for the pathogenesis of idiopathic nephrotic syndrome are more likely to be immature differentiating cells rather than mature peripheral T cells.

Klf6 is a zinc finger protein expressed in a cell-specific manner during kidney development.

Molecular mechanisms that are responsible for the development of the renal collecting duct system during embryogenesis are still poorly understood. A mouse cDNA encoding a zinc finger protein, called Klf6, which is a member of the Krüppel-like family of transcription factors, has been cloned. Northern blot analyses showed that Klf6 was already expressed in 11.5-d postconception mouse embryos and that its expression persisted after birth. They also disclosed that Klf6 had a restricted pattern of expression. In situ hybridization experiments using mouse embryos showed that during kidney development, Klf6 was expressed selectively in the Wolffian duct and in its derivatives. During mesonephros development, it was expressed in the Wolffian duct but not in the mesonephric mesenchyme. Thereafter, Klf6 was expressed in the ureteric bud and its branches and in the collecting ducts, whereas it was not expressed in tubular structures that derive from the metanephric mesenchyme. Glomeruli were not labeled during early stages of differentiation, and it is only at the capillary stage that a staining of the mesangial area was observed, which persisted after birth. This pattern of expression is strikingly similar to the one of GATA-3, which is another zinc finger protein. It suggests that Klf6 may play a role during kidney development and in particular during the development of the renal collecting duct system, possibly in association with GATA-3.